Nagatomo Shigenori, Okumura Miki, Saito Kazuya, Ogura Takashi, Kitagawa Teizo, Nagai Masako
Department of Chemistry, Faculty of Pure and Applied Sciences, University of Tsukuba , Tsukuba, Ibaraki 305-8571, Japan.
Picobiology Institute, Graduate School of Life Science, University of Hyogo , 3-2-1 Kouto, Kamigori, Ako-gun, Hyogo 678-1297, Japan.
Biochemistry. 2017 Mar 7;56(9):1261-1273. doi: 10.1021/acs.biochem.6b01118. Epub 2017 Feb 24.
Regulation of the oxygen affinity of human adult hemoglobin (Hb A) at high pH, known as the alkaline Bohr effect, is essential for its physiological function. In this study, structural mechanisms of the alkaline Bohr effect and pH-dependent O affinity changes were investigated via H nuclear magnetic resonance and visible and UV resonance Raman spectra of mutant Hbs, Hb M Iwate (αH87Y) and Hb M Boston (αH58Y). It was found that even though the binding of O to the α subunits is forbidden in the mutant Hbs, the O affinity was higher at alkaline pH than at neutral pH, and concomitantly, the Fe-His stretching frequency of the β subunits was shifted to higher values. Thus, it was confirmed for the β subunits that the stronger the Fe-His bond, the higher the O affinity. It was found in this study that the quaternary structure of α(Fe)β(Fe-CO) of the mutant Hb is closer to T than to the ordinary R at neutral pH. The retained Aspβ94-Hisβ146 hydrogen bond makes the extent of proton release smaller upon ligand binding from Hisβ146, known as one of residues contributing to the alkaline Bohr effect. For these T structures, the Aspα94-Trpβ37 hydrogen bond in the hinge region and the Tyrα42-Aspβ99 hydrogen bond in the switch region of the α-β interface are maintained but elongated at alkaline pH. Thus, a decrease in tension in the Fe-His bond of the β subunits at alkaline pH causes a substantial increase in the change in global structure upon binding of CO to the β subunit.
成人血红蛋白（Hb A）在高pH值下的氧亲和力调节，即所谓的碱性玻尔效应，对其生理功能至关重要。在本研究中，通过突变型血红蛋白Hb M岩手（αH87Y）和Hb M波士顿（αH58Y）的氢核磁共振以及可见和紫外共振拉曼光谱，研究了碱性玻尔效应和pH依赖性氧亲和力变化的结构机制。研究发现，尽管在突变型血红蛋白中氧与α亚基的结合被禁止，但在碱性pH值下氧亲和力高于中性pH值，同时，β亚基的铁-组氨酸伸缩频率向更高值移动。因此，对于β亚基而言，铁-组氨酸键越强，氧亲和力越高这一点得到了证实。本研究发现，在中性pH值下，突变型血红蛋白的α(Fe)β(Fe-CO)四级结构更接近T态而非普通的R态。保留的Aspβ94-Hisβ146氢键使得配体与Hisβ146结合时质子释放程度较小，Hisβ146是导致碱性玻尔效应的残基之一。对于这些T态结构，α-β界面铰链区的Aspα94-Trpβ37氢键和开关区的Tyrα42-Aspβ99氢键得以维持，但在碱性pH值下伸长。因此，碱性pH值下β亚基铁-组氨酸键张力的降低导致CO与β亚基结合后整体结构变化大幅增加。
